1. Field of the Invention
The present invention is related to biotechnology, specifically to a method for producing L-amino acids by fermentation, and more specifically to a gene obtained from Escherichia coli. This gene is useful for improving L-amino acid productivity of the bacterium, for example, L-phenylalanine and L-threonine.
2. Brief Description of the Related Art
Conventionally, L-amino acids have been industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants of these strains which have been specifically modified to enhance L-amino acid productivity.
Many techniques for enhancing L-amino acid productivity have been disclosed, for example, by transformation of a microorganism with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). These techniques are based on increasing the activities of the enzymes involved in amino acid biosynthesis and/or desensitizing the enzymes subject to feedback inhibition by the L-amino acid (see, for example, Japanese Laid-open application No. 56-18596 (1981), WO 95/16042, or U.S. Pat. Nos. 5,661,012 and 6,040,160).
Alternatively, enhancing amino acid secretion may improve the productivity of a strain which can produce L-amino acids. Corynebacterium, which produce lysine and have increased expression of the L-lysine excretion gene (lysE gene), have been disclosed (WO 9723597A2). In addition, genes encoding efflux proteins suitable for secretion of L-cysteine, L-cystine, N-acetylserine or thiazolidine derivatives have also been disclosed (U.S. Pat. No. 5,972,663).
Several Escherichia coli genes have been disclosed which encode putative membrane proteins which function to increase L-amino acid production. The presence of additional copies of the rhtB gene cause a bacterium to become more resistant to L-homoserine, and therefore increase the production of L-homoserine, L-threonine, L-alanine, L-valine, and L-isoleucine by the bacterium (European patent application EP994190A2). The presence of additional copies of the rhtC gene cause a bacterium to become more resistant to L-homoserine and L-threonine, and therefore increase production of L-homoserine, L-threonine, and L-leucine (European patent application EP1013765A1). The presence of additional copies of the yahN, yeaS, yfiK, and yggA genes increase production of L-glutamic acid, L-lysine, L-threonine, L-alanine, L-histidine, L-proline, L-arginine, L-valine, and L-isoleucine (European patent application EP1016710A2).
The present inventors made a thrR mutant of E.coli K-12, also referred to as rhtA23, that displays resistance to high concentrations of threonine or homoserine in a minimal medium (Astaurova, O. B. et al., Appl. Biochem. Microbiol., 21, 611-616,1985). This mutation improved the production of L-threonine (SU Patent No. 974817), homoserine, and glutamate (Astaurova, O. B. et al., Appl. Biochem. Microbiol., 27, 556-561, 1991) by their respective E. coli producing strains.
Furthermore, the present inventors have reported that the rhtA gene is present at 18 min on the E.coli chromosome. This location is close to the glnHPQ operon that encodes components of the glutamine transport system. Also, the rhtA gene is identical to the ybiF ORF, which is located between the pexB and ompX genes. The DNA sequence expressing a protein encoded by this ORF has been designated the rhtA (rht: resistance to homoserine and threonine) gene.
The present inventors have also found that amplification of the rhtA gene confers resistance to homoserine and threonine. The rhtA23 mutation is an A-for-G substitution at position −1, with respect to the ATG start codon (ABSTRACTS of 17th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, Calif. Aug. 24-29, 1997, abstract No. 457).
It is known that the nucleotide sequence of the spacer between the SD sequence and start codon, and especially the sequences immediately upstream of the start codon, profoundly affect mRNA translatability. A 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al., Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al., EMBO J., 3, 623-629, 1984). Therefore, these observations suggest that the rhtA23 mutation increases rhtA gene expression.
The rhtA gene encodes a protein of 295 amino acid residues and is a highly hydrophobic. There are 10 predicted transmembrane regions. A PSI-BLAST search of the nucleotide sequence of E.coli strain K-12 among the genus Escherichia (Science, 277, 1453-1474 (1997) revealed at least 10 proteins homologous to RhtA. Among these are proteins encoded by the ydeD and yedA genes. The ydeD gene is known to be involved in the efflux of cysteine pathway metabolites (Dasler et al., Mol. Microbiol., 36, 1101-1112, 2000; U.S. Pat. No. 5,972,663). The yedA gene is known as a putative transmembrane subunit, which encodes a protein for which the function is unknown (numbers 8037 to 8957 in the sequence of GenBank accession AE000287 U00096).